Method for treating biomass for producing cell lysate containing plasmid DNA

ABSTRACT

The method of making the clear cell lysate containing plasmid DNA from a biomass obtained in a cell culture process includes filtering the biomass obtained in the cell culture process with a filter medium in the presence of diatomaceous earth acting as a filtering agent to form a filter cake containing the biomass and the filter medium, then thermally digesting the biomass in the filter cake to form a cell lysate containing the plasmid DNA and filtering the cell lysate to form a clear filtrate. The clear filtrate contains the plasmid DNA and has a clarity characterized by an OD 600  of at maximum 0.05 U/cm.

BACKGROUND OF THE INVENTION

The present invention relates to a method for the integrated treatmentof biomass from a cell culture process for producing clear cell lysatecontaining plasmid DNA, and a plasmid DNA cell lysate produced by themethod.

Treatment is to be understood as meaning a conditioning method forproviding clear cell lysate containing plasmid DNA. An integrated methodis to be understood as meaning a method in which the individual steps ofthe method are contiguous with one another so that the product stream isconveyed virtually continuously. The basically multistage method can becarried out continuously or batchwise.

The separation of biological material from a cell culture process, thedigestion of the biological material and the production of a clear celllysate containing plasmid DNA is a customary method in the area ofmolecular biology. In the known methods, biological material, forexample comprising E. coli bacteria cells, is separated from the culturesupernatant, resuspended and then digested. Separating off the solidconstituents gives a clear cell lysate which contains the plasmid DNA inaddition to genomic DNA, RNA, proteins and endotoxins.

It is known that biological materials can be separated from the cellculture process by batchwise or continuous centrifuging. The separationof baker's yeast cells by filtration over a bed of filtering agent isdescribed in GB-A-1082862. Said patent also discloses the separation ofcell residues from a yeast autolysate by means of a filtering agent.

The customary methods for cell digestion are the known alkaline lysisand thermal lysis. In order to improve the cell digestion, enzymes, forexample lysozyme, and/or detergents are frequently added to the cellsuspension. In the alkaline lysis method, a precipitate whichsubstantially contains the cell debris and parts of the genomic DNA andof the protein is obtained after the cell digestion at pH 12 by additionof sodium hydroxide solution and sodium dodecylsulphate and subsequentneutralization with a high molecular weight acetate buffer. The completeisolation of this precipitate can be achieved only by thoroughcentrifuging. A centrifugal acceleration of 12000 g is not sufficientfor this purpose (I. Feliciello et al., Anal. Biochem. 212 (1993)394-401). The precipitate can be very substantially isolated only at acentrifugal acceleration of 26000 g for 30 minutes at 4° C. On the otherhand, the filtration of this precipitate is associated with low processrates and large losses of plasmid DNA, even with the aid of very finefiltering agents or in combination with a flotation step. The adsorptionof plasmid DNA on the surface of the filtering agent contributesconsiderably to the losses. In particular, plasmid DNA binds veryrapidly and strongly to such mineral surfaces if the concentration ofdivalent cations exceeds 0.1 mmol or the monovalent cations exceed 20mmol in the case of potassium or 50 mmol in the case of sodium (G.Romanowski et al., Appl. Environ. Microbiol. 57 (1991) 1057-1061).

High molecular weight nucleic acids are sensitive to hear forces. Strongshear forces can lead to irreversible damage to nucleic acids, inparticular to breaks in the strand. For this reason, mechanicaldigestion methods are seldom used for nucleic acid treatment (A. Carlsonet al., Biotechnol. Bioeng. 48 (1995) 303-315). It is also known that,in industrial centrifuges with continuous introduction of liquid, highshear forces at the rotor inlet act on the nucleic acids and inevitablylead to breaks in the strand.

WO 96/36706 describes a method in which microorganisms are digested inthe presence of detergents (Triton®) and by heating to 70° C. to 1000°C. in a flow-through heat exchanger. This is a combination methodcomprising thermolysis and with a detergent (optionally with lysozyme).In the absence of lysozyme, a thermal treatment for 30 seconds isdescribed; in the presence of lysozyme, a thermal treatment for 6seconds is described. On cell digestion by heating, solid compoundscomprising debris, genomic DNA and proteins form. In addition, verysubstantial denaturing of DNA-degrading enzymes, so-called DNases, maybe assumed as a result of the heat treatment. A clear cell lysate isobtained after batchwise centrifuging. In this method, only the celldigestion is carried out continuously, the solid constituents beingseparated off batchwise by centrifuging. In order to obtain the finalclear cell lysate, a filtration over a membrane filter also had to becarried out after the centrifuging.

According to WO 92/207863 an apparatus and a method for isolation ofnucleic acids from cell suspensions are known. According to this, thecells are immobilized in the cavities of an upstream, porous matrix inthe form of a layer. This is achieved by deep filtration in the matrix,by virtue of the fact that the cavity size is of the order of magnitudeof the cells and the matrix surface has ion exchange properties. Theparticle size of the matrix is 10 to 50 μm. Owing to the ion exchangeproperties, DNA is adsorbed on the matrix surface. The invention doesnot relate to adsorption of DNA. The known method gives purified DNA butnot a clear cell lysate containing plasmid DNA.

EP-A-0814156 describes a method for purifying DNA. The kieselguhrs usedtherein are not specified. The lysis is affected by the alkaline method.Thermal lysis is not described.

The prior art thus discloses neither methods which make it possible towork up large amounts of biomass in an integrated manner nor methods bymeans of which large amounts of clear cell lysates containing plasmidDNA can be provided by an integrated procedure.

There is a need for an efficient conditioning method for providing clearcell lysate containing plasmid DNA from a cell culture process whichpermits plasmid DNA purification.

SUMMARY OF THE INVENTION

It is therefore the object of the present invention to provide anintegrated working-up method which makes it possible to provide largeamounts of clear cell lysates with high plasmid DNA yield from a biomassin an efficient and controlled manner while avoiding the disadvantagesof the alkaline lysis method and of centrifuging.

This object is achieved, according to the invention, if in each casefiltration is affected in a first stage in a filter in the presence of afiltering agent, the biomass contained in the filter cake is thermallydigested in a second stage and the cell lysate is filtered in a furtherstage.

It has now been found that any volume from 50 ml to 10000 l of biomasscan be rapidly processed to give clear cell lysate containing theplasmid DNA by the method of the present invention. Particularlyadvantageous is the fact that, in the method of the present invention,the applied excess pressure during filtration provides a check withregard to the shear forces acting on the nucleic acids. In particular,after separation by gel electrophoresis, a cell lysate producedaccording to the invention clearly shows the bands for plasmid DNA, inaddition to the bands for genomic DNA and RNA, without detectable damageto the nucleic acids due to breaks in the strand. Moreover, the celllysate obtained is freed from unpleasant odours by the heat treatment.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is expedient that the filtering agent is present during thedigestion. This has the advantage that the filtering agent required forthe subsequent clarifying filtration of the cell lysate is alreadypresent. The continuous use of the same filtering agent over all steps(isolation, digestion, clarification) is also efficient in terms ofprocess engineering and therefore economical. It is particularlyadvantageous if, instead of suspending the filter cake for the lysis,combined thermal digestion and clarifying filtration are carried outdirectly in the existing filter cake (for example by pumping through hotlysis buffer) and thus very clear cell lysate can be obtained directly.Consequently, the entire method is more efficient and hence economicallymore advantageous as a result of the reduction of the number ofindividual steps.

The choice of the filtering agent is of decisive importance. Thefiltering agent must be inert in order to minimize adsorption effects.It has proved expedient to use, as the filtering agent, calcinedkieselguhrs having an SiO₂ content of at least 90% by weight andadditionally having the following properties:

-   -   Wet density: 0.2-0.4 g/cm³    -   Permeability: 0.02-2 Darcy    -   BET surface area: 2-20 m²/g    -   Geometrical surface area :0.25-0.65 m²/g    -   Particle size: 2.5-8.0 μm    -   Particle size retention (99%): 0.1-2 μm

This has the advantage that very clear filtrate having a sufficientlyhigh filtration flow rate at moderate differential pressure is obtainedin one filtration step. In addition to this combination of efficiencyand effectiveness, the use of highly pure filtering agents has theadvantage that their surfaces are standardized and hence theirproperties can be controlled.

It is also expedient that the same filtering agent is used in allstages. This has the advantage that the individual steps of the methodare contiguous with one another so that the product stream is conveyedvirtually continuously.

The amount of filtering agent, based on the solids content of thesolution to be filtered, the filtration area and the level of themaximum excess filtration pressure to be applied can only be determinedempirically.

It has proved particularly expedient to carry out the digestionthermally. This has the advantage that the digestion can be carried outunder mild conditions, for example in the physiological pH range, undervery readily controllable and reproducible conditions. The process ratecan be considerably increased if a flow-through heat exchanger is usedinstead of the batchwise heating of the cell suspension. It may also beassumed that a part of the DNA-degrading enzyme is deactivated onheating. In comparison with the alkaline lysis, the odour annoyance inthermal lysis is substantially lower.

The digestion is carried out at temperatures between 70° C. and 90° C.,preferably at 70° C. to 85° C., in particular at 80° C. At a temperatureof less than 70° C., the thermal digestion is incomplete; at atemperature above 90° C., plasmid DNA melts. The duration of the thermaltreatment can only be determined empirically.

It is 30 seconds to a few minutes.

The digestion must be carried out at a pH between 7 and 10. In this pHrange, the adsorption losses of plasmid DNA are minimal and the activityof lysozyme is optimal. Moreover, plasmid DNA is already present in thephysiological pH range in the clear cell lysate obtained.

It is expedient to carry out the digestion in the presence ofexclusively monovalent cations. This has the advantage that losses inthe final filtration to give the clear plasmid DNA lysate owing toadsorption on the surface of the filtering agent are minimal. Polyvalentcations which are liberated during the cell digestion are masked bycomplexing agents.

The maximum concentration of cations should be not more than 20 mmol forK⁺, not more than 50 mmol for Na⁺ or not more than 150 mmol for NH₄ ⁺.When these maximum concentrations are exceeded, losses of plasmid DNAoccur owing to adsorption on the surface of the filtering agent. If aplurality of cation species are present, the additive nature of theeffect must be taken into account when specifying the permissible totalconcentration.

Clear cell lysate containing plasmid DNA is distinguished by a clarityOD₆₀₀ of not more than 0.05 U/cm, corresponding to a decrease in theturbidity of at least 99%. The resulting clear cell lysate with theplasmid DNA can be used for cloning, for transformation, fortransfection, for microinjection into cells, for gene therapy, for DNAvaccination and/or for polymerase chain reaction (PCR).

The invention is to be described in more detail with reference toexamples.

EXAMPLES Example 1

Plasmid DNA pHEN1 (4618 bp) in the E. coli strain TG1 was multiplied in2TY medium at 37° C. for 16 hours in a shaken flask. 15, 30 or 60 g/l ofCelpureP300® (trade mark of World Minerals Inc.) were added to three E.coli suspensions of 125 ml each (OD₆₀₀=6) and stirring was carried outcontinuously at 20° C. Each suspension was then transferred to a suctionfilter (10 cm² filter area, 200 ml capacity) in which a prefilter cakeof 2 kg/m² of CelpureP3000® had been built up beforehand over a filtermedium comprising polypropylene monofilament (pore size 10 μm). Thefiltration was carried out at an excess pressure of 0.5 bar and 20° C.

In all cases, a clear filtrate was obtained. The highest mean filtrationflow rate (2.8 m³/m² h) was achieved with 30 g/l of CelpureP300®. Theresults are summarized in Table 1.

TABLE 1 Mean filtration OD₆₀₀ of the Decrease in CelpureP300 ®(*) flowrate filtrate turbidity in in g/l (m³/m²h) (U/cm) % 60 1.8 0.04 99 302.8 0.05 99 15 2.4 0.03 99 (*)Properties: Silica content: 98.65%; wetdensity: 0.26 g/cm³. Permeability: 0.15-0.30 Darcy; BET surface area:4-6 m²/g; geometrical surface area: 0.62 m²/g; particle size retention(99%): 0.6-0.75 μm.

Example 2

Lysis Buffer A:

150 mmol of Tris.HCl, 25 mmol of Na₂EDTA, 8% by weight of sucrose (pH 9)

Lysis buffer B:

As for lysis buffer A, but additionally 2% of TritonX-100® (trade markof Rohm und Haas)

Two filter cakes from Example 1 with 30 g/l of CelpureP300® were eachresuspended in 190 ml of lysis buffer A or B. 500 U/ml of lysozyme wereadded in each case and the suspensions were heated to 80° C. for 30seconds with continuous stirring. The still hot suspensions weretransferred to a suction filter in which a prefilter cake of 2 kg/m² ofCelpureP300® (incubated for 15 minutes in the corresponding lysisbuffer) had been built up over a filter medium comprising polypropylenemonofilament (pore size 10 μm). The filtration was carried out at 0.5bar excess pressure.

By means of the lysate buffer A, a clear cell lysate (OD₆₀₀=0.02) with amean filtration flow rate of 0.4 m³/m² h was obtained. In contrast, themean filtration flow rate was reduced by 50% by the addition of TritonX-100® (lysis buffer B). A significant increase in the mean filtrationflow rate to 1.5 m³/m² h in the lysis buffer A was achieved when thetotal amount of CelpureP300® ad been adjusted to 100 g/l and when PVDFwarp/PTFE weft monofilament (pore size 11.5 μm) had been used as thefilter medium.

Example 3

3.7 ml of λ DNA (E. coli; 48.5 kb dsDNA; 0.51 mg/ml) were added to 25 mlof a buffer solution consisting of 250 mmol of Tris.HCl, 25 mmol ofNa₂EDTA and 8% by weight of sucrose (pH 9.09). 0.75 g of CelpureP300®(30 g/l) was added and the suspension was shaken for 30 minutes at 20°C. and 200 rpm.

UV absorption measurements at 260 nm and a PicoGreen® test (kit fromMolekular Probes Inc.) showed no difference between the λ DNAconcentration in the supernatant of the suspension and the concentrationin the starting solution.

Example 4

E. coli cells DH5α with the plasmid DNA pEGFP-N1 were divided into fourequal parts and each part suspended in a buffer consisting of Tris.HCland 10 mM EDTA. From one of these suspensions, the cells were digestedusing the Nucleobond® AX kit (trade mark of Macherey-Nagel) by thealkaline lysis method, and the plasmid DNA was isolated and purified. Inthe case of the other three suspensions, the cells were thermallydigested for 5 minutes at 70° C./pH 9, 70° C./pH 10 and 60° C./pH 10,and the plasmid DNA was isolated and purified in each case using thesame kit. The plasmid DNA quantification of the four samples by means ofthe PicoGreen test showed that the digestion by the alkaline lysismethod and the thermal lysis at 70° C. and pH 9 or pH 10 led to the sameyields of the plasmid DNA, whereas the thermal digestion at 60° C. andpH 10 gave only about 30% of the yield of the plasmid DNA obtained bythe other three methods. From Example 3, it is known that no adsorptionof the plasmid DNA on the surface of the filtering agent is to beexpected under the thermal lysis conditions described.

Method of Measurement:

-   -   Clarity Measurement    -   The clarity of a filtrate (optical density, OD) was determined        using a Lambda 20 UV/Vis spectrometer from Perkin-Elmer in the        absorption mode at 600 nm and a wavelength of 1 cm against water        as reference at room temperature.

1. A method of making a clear cell lysate containing plasmid DNA from abiomass obtained in a cell culture process, said method consisting ofthe steps of: a) suspending the biomass together with a filtering agentto form a first suspension, wherein the filtering agent is a kieselguhrand said kieselguhr contains greater than or equal to 90% silica, a wetdensity of 0.2 to 0.4 g/cm³, a permeability of 0.02 Darcy, a BET surfacearea of 2 to 20 m²/g, a geometrical surface area of 0.25 to 0.65 m²/g ,a particle size of 2.5 to 8.0 μm, and a 99% particle size retention of0.1 to 0.2 μm; b) filtering the first suspension in the presence of thefiltering agent with a filter medium to form a filter cake containingsaid biomass; c) after the filtering of step b), re-suspending thefilter cake formed during said suspending in a lysis buffer at pH 7 to10 to form a second suspension, wherein the lysis buffer consists of 150mmol of Tris-HCL, 25 mmol of Na₂EDTA, 8% by weight of sucrose, andoptionally containing 2% TritonX-100®; d) heating said second suspensionat temperatures between 70° C. to 90° C. for at least 30 seconds tothermally digest said biomass; e) after the heating of step d),filtering said second suspension with the filter medium in the presenceof the filtering agent to obtain the cell lysate; wherein said filteringof said second suspension and said first suspension consists offiltering only with the aid of vacuum or pressure; f) filtering the celllysate to form a clear filtrate containing said plasmid DNA and having aclarity OD₆₀₀ of not more than 0.05 U/cm, which corresponds to adecrease of turbidity of at least 99%, and in which said method does notinclude centrifugation.
 2. The method as defined in claim 1, whereinsaid filter agent is diatomaceous earth.
 3. A method of making a clearcell lysate containing plasmid DNA from a biomass obtained in a cellculture process, said method consisting of the steps of: a) suspendingthe biomass together with a filtering agent to form a first suspension,wherein the filtering agent is a kieselguhr and said kieselguhr containsgreater than or equal to 90% silica, a wet density of 0.2 to 0.4 g/cm³,a permeability of 0.02 Darcy, a BET surface area of 2 to 20 m²/g, ageometrical surface area of 0.25 to 0.65 m²/g , a particle size of 2.5to 8.0 μm, and a 99% particle size retention of 0.1 to 0.2 μm; b)filtering the first suspension in the presence of the filtering agentwith a filter medium to form a filter cake containing said biomass; andc) pumping a hot lysis buffer having a pH of 7 to 10 and a temperatureof from 70° C. to 90° C. through said filter cake in order to thermallydigest said biomass contained in said filter cake to form a cell lysateand to perform a clarification filtration of the cell lysatesimultaneously to obtain a clear filtrate, said clear filtratecontaining said plasmid DNA and having a clarity OD₆₀₀ of not more than0.05 U/cm, which corresponds to a decrease of turbidity of at least 99%,wherein the lysis buffer consists of 150 mmol of Tris-HCL, 25 mmol ofNa₂EDTA, 8% by weight of sucrose, and optionally containing 2%TritonX-100®, and in which said method does not include centrifugation.4. The method as defined in claim 3, wherein said filter agent isdiatomaceous earth.